The RecQ helicase family is a group of highly conserved DNA unwinding enzymes described as caretakers of the genome. In humans, loss of function of three of the five members of the RecQ family (RECQ1, WRN, BLM, RECQL4, and RECQL5) is genetically linked with rare cancer predisposition disease (Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome). Our goal is to determine common and specialized functions of human RecQ helicases in mechanisms of genome maintenance. The overall focus is on elucidating how impaired function of a specific RecQ protein relates to disease outcomes, including cancer predisposition and premature aging. Loss of RECQ1, the most abundant RecQ homolog in humans, is sufficient to cause genomic instability in mouse and human cells. Work in current funding period identified novel interactions of RECQ1 with proteins that support a role in mechanisms of DNA strand break repair. Furthermore, RECQ1 was enriched at genomic loci that are intrinsically difficult to replicate due to their propensity o form secondary structures. Non-B DNA structures including G4 DNA may present challenges to both replication and transcription. RECQ1 alters gene expression, in part, through its specific binding with G4 motifs predicted to form G4 DNA structures in the target gene promoters in vivo. In this renewal application, we hypothesize that RECQ1 helicase facilitates genome maintenance mechanisms of DNA repair and transcriptional regulation through specific protein partners and recognition of specialized DNA structures. Our Specific Aims are: to determine the role of RECQ1 in genome maintenance through specific protein partners and chromatin interactions; and to identify RecQ-regulated transcriptome in isogenic background and determine its significance in RecQ functions. Knowledge gained through the proposed study will provide essential framework for exploring in more detail the specific roles of RECQ1 in genome maintenance and its broader significance in cellular homeostasis. Given the importance of genome maintenance as a mechanism to prevent cancer and other genetic diseases, understanding how the loss of a specific RecQ protein may promote genomic instability and cancer susceptibility characteristic of the heritable disease it causes is essential to uncover how individual RecQ homologs work in context of the human health.